Arsenic enters human body from both geological and anthropogenic sources. Because of the ubiquity of arsenic in the environment, every organism has developed transport systems for the efflux and detoxification of arsenic. Chronic exposure to arsenic has been linked to cardiovascular and peripheral vascular diseases, neurological disorders, diabetes and various cancers. Arsenic-containing drugs are used as chemotherapeutic agents for the treatment of leukemia and parasitic diseases. An understanding of both arsenic chemistry and the molecular details of arsenic transport systems is essential for alleviating the problems of arsenic toxicity, as well as for the rational design of drugs to treat drug-resistant microbes and cancer cells. We have identified aquaglyceroporins as a major pathway for trivalent arsenical [As(lll)] uptake in organisms from E. coli to humans. We have also shown that As(lll) is transported by hexose permeases in yeast and humans. The overall goal of this proposal is elucidation of the molecular mechanisms of arsenic transport in microorganisms. The presence of arsenic resistance (ars) genes in the genome of every living organism sequenced to date illustrates first that ars genes must be ancient and second that arsenic must still be ubiquitous in the environment, providing the selective pressure that maintains them in present-day organisms. The first specific aim proposes to study the mechanisms of uptake of trivalent arsenic by aquaglyceroporins and hexose permeases in two bacteria (E. coli and Sinorhizobium meliloti), and, for comparative purposes, in the eukaryotic microorganism Saccharomyces cerevisiae. In particular, the mechanism of substrate selectivity that allows arsenic transport will be determined. The second specific aim is a detailed structure-function analysis of the ArsAB As(lll)-translocating ATPase, the best-characterized detoxification system for trivalent arenic. This arsenic extrusion pump is encoded by the arsenical resistance operon of the clinically isolated resistance plasmid R773. The project entails detailed molecular analysis of the nucleotide binding domains, metal binding sites and signal transduction domains of the ArsA ATPase, as well as the way in which ArsA interacts with ArsB, the membrane component of the pump. Finally, the interaction of the new and novel arsenic chaperone protein, ArsD, with the ArsAB pump will be studied.